Device for automated detection of nucleic acids

ABSTRACT

The present invention relates to the detection of specific nucleic acid sequences after an amplification process, or directly without amplification. In particular, the invention provides for the automation of the amplification and detection process, the amplification and detection of one or more specific nucleic acid sequences, the use of internal controls, reduced potential for contamination caused by the manual manipulation of reagents, and improved reagent compositions to better control assay performance and provide for further protection against contamination.

This application is a divisional application of U.S. patent applicationSer. No. 08/850,171, filed May 2, 1997, now abandoned.

FIELD OF THE INVENTION

The present invention relates to the detection of specific nucleic acidsequences in a target test sample.

In particular, the present invention relates to the automated detectionof specific nucleic acid sequences which are either unamplified oramplified nucleic acid sequences (amplicons).

In addition, the present invention relates to the use of automatedamplification, methods and compositions for monitoring successfulamplification, improved methods for reducing the chance forcontamination, and the use of unified reaction buffers and unit dosealiquots of reaction components for amplification.

Finally, the present invention also relates to unique constructs andmethods for the conventional or automated detection of one, or more thanone different nucleic acid sequences in a single assay.

THE BACKGROUND OF THE INVENTION

The development of techniques for the manipulation of nucleic acids, theamplification of such nucleic acids when necessary, and the subsequentdetection of specific sequences of nucleic acids or amplicons hasgenerated extremely sensitive and nucleic acid sequence specific assaysfor the diagnosis of disease and/or identification of pathogenicorganisms in a test sample.

Amplification of Nucleic Acids

When necessary, enzymatic amplification of nucleic acid sequences willenhance the ability to detect such nucleic acid sequences. Generally,the currently known amplification schemes can be broadly grouped intotwo classes based on whether, the enzymatic amplification reactions aredriven by continuous cycling of the temperature between the denaturationtemperature, the primer annealing temperature, and the amplicon (productof enzymatic amplification of nucleic acid) synthesis temperature, orwhether the temperature is kept constant throughout the enzymaticamplification process (isothermal amplification). Typical cyclingnucleic acid amplification technologies (thermocycling) are polymerasechain reaction (PCR), and ligase chain reaction (LCR). Specificprotocols for such reactions are discussed in, for example, ShortProtocols in Molecular Biolog, 2^(nd) Edition, A Compendium of Methodsfrom Current Protocols in Molecular Biology, (Eds. Ausubel et al., JohnWiley & Sons, New York, 1992) chapter 15. Reactions which are isothermalinclude: transcription-mediated amplification (TMA), nucleic acidsequence-based amplification (NASBA), and strand displacementamplification (SDA).

U.S. Patent documents which discuss nucleic acid amplification includeU.S. Pat. Nos. 4,683,195; 4,683,202; 5,130,238; 4,876,187; 5,030,557;5,399,491; 5,409,818; 5,485,184; 5,409,818; 5,554,517; 5,437,990 and5,554,516 (each of which are hereby incorporated by reference in theirentirety). It is well known that methods such as those described inthese patents permit the amplification and detection of nucleic acidswithout requiring cloning, and are responsible for the most sensitiveassays for nucleic acid sequences. However, it is equally wellrecognized that along with the sensitivity of detection possible withnucleic acid amplification, the ease of contamination by minute amountsof unwanted exogenous nucleic acid sequences is extremely great.Contamination by unwanted exogenous DNA or RNA nucleic acids is equallylikely. The utility of amplification reactions will be enhanced bymethods to control the introduction of unwanted exogenous nucleic acidsand other contaminants.

Prior to the discovery of thermostable enzymes, methods that usedthermocycling were made extremely difficult by the requirement for theaddition of fresh enzyme after each denaturation step, since initiallythe elevated temperatures required for denaturation also inactivated thepolymerases. Once thermostable enzymes were discovered, cycling nucleicacid amplification became a far more simplified procedure where theaddition of enzyme was only needed at the beginning of the reaction.Thus reaction tubes did not need to be opened and new enzyme did notneed to be added during the reaction, allowed for an improvement inefficiency and accuracy as the risk of contamination was reduced, andthe cost of enzymes was also reduced. An example of a thermostableenzyme is the polymerase isolated from the organism Thermophilusaquaticus.

In general, isothermal amplification can require the combined activityof multiple enzyme activities for which no optimal thermostable variantshave been described. The initial step of an amplification reaction willusually require denaturation of the nucleic acid target, for example inthe TMA reaction, the initial denaturation step is usually ≧65° C., butcan be typically ≧95° C., and is used when required to remove thesecondary structure of the target nucleic acid.

The reaction mixture is then cooled to a lower temperature which allowsfor primer annealing, and is the optimal reaction temperature for thecombined activities of the amplification enzymes. For example, in TMAthe enzymes are generally a T7 RNA polymerase and a reversetranscriptase (which includes endogenous RNase H activity). Thetemperature of the reaction is kept constant through out the subsequentisothermal amplification cycle.

Because of the lack of suitable thermostable enzymes, some isothermalamplifications will generally require the addition of enzymes to thereaction mixture after denaturation at high temperature, and cool-downto a lower temperature. This requirement is inconvenient, and requiresthe opening of the amplification reaction tube, which introduces a majoropportunity for contamination.

Thus, it would be most useful if such reactions could be more easilyperformed with a reduced risk of contamination by methods which wouldallow for integrated denaturation and amplification without the need formanual enzyme transfer.

Amplification Buffer and Single Reaction Aliquot of Reagents

Typical reaction protocols require the use of several different buffers,tailored to optimize the activity of the particular enzyme being used atcertain steps in the reaction, or for optimal resuspension of reactioncomponents. For example, while a typical PCR 10×amplification bufferwill contain 500 mM KCl and 100 mM Tris HCl, pH 8.4, the concentrationof MgCl₂ will depend upon the nucleic acid target sequence and primerset of interest. Reverse transcription buffer (5×) typically contains400 mM Tris-Cl, pH 8.2; 400 mM KCl and 300 mM MgCl₂, whereas MurineMaloney Leukemia Virus reverse transcriptase buffer (5×) typicallycontains 250 mM Tris-Cl, pH 8.3; 375 mM KCl; 50 mM DTT (Dithiothreitol)and 15 mM MgCl₂.

While such reaction buffers can be prepared in bulk from stockchemicals, most commercially available amplification products providebulk packaged reagents and specific buffers for use with theamplification protocol. For example, commercially available manualamplification assays for detection of clinically significant pathogens(for example Gen-Probe Inc. Chlamydia, and Mycobacterium tuberculosisdetection assays) requires several manual manipulations to perform theassay, including dilution of the test sample in a sample dilution buffer(SDB), combination of the diluted sample with amplification reactionreagents such as oligonucleotides and specific oligonucleotidepromoter/primers which have been reconstituted in an amplificationreconstitution buffer (ARB), and finally, the addition to this reactionmixture of enzymes reconstituted in an enzyme dilution buffer (EDB).

The preparation and use of multiple buffers which requires multiplemanual additions to the reaction mixture introduces a greater chance forcontamination. It would be most useful to have a single unified bufferwhich could be used in all phases of an amplification protocol. Inparticular, with the commercially available TMA assays described above,the requirement for three buffers greatly complicates automation of sucha protocol.

Bulk packaging of the enzyme or other reaction components bymanufacturers, may require reconstitution of the components in largequantities, and the use of stock amounts of multiple reagents, can bewasteful when less than the maximal number of reactions are to becarried out, as some of these components may be stable for only a shorttime. This process of reconstitution also requires multiplemanipulations by the user of the stock reagents, and aliquoting ofindividual reaction amounts of reagents from stocks which creates amajor opportunity for contamination.

Methods and compositions for the preparation of bulk quantities ofpreserved proteins are known, see for example, U.S. Pat. Nos. 5,098,893;4,762,857; 4,457,916; 4,891,319; 5,026,566 and International PatentPublications WO 89/06542; WO 93/00806; WO 95/33488 and WO 89/00012, allof which are hereby incorporated by reference in their entirety.However, the use of pre-aliquoted and preserved reagent components insingle reaction quantities/dose is both very useful and economical.Single aliquots of enzyme reagent avoids multiple use of bulk reagents,reducing waste, and greatly reducing the chance of contamination.Further, such single reaction aliquots are most suitable for theautomation of the reaction process.

The requirement for many changes of buffer and the multiple addition ofreagents complicates the automation of such reactions. A single doseunit of reaction buffer mixture, and a unified combination buffer willboth simplify automation of the process and reduce the chance ofcontamination.

Automation of nucleic Acid Detection with or without Amplification

Nucleic acid probe assays, and combination amplification/probe assayscan be rapid, sensitive, highly specific, and usually require precisehandling in order to minimize contamination with non-specific nucleicacids, and are thus prime candidates for automation. As withconventional nucleic acid detection protocols, it is generally requiredto utilize a detection probe oligonucleotide sequence which is linked bysome means to a detectable signal generating component. One possibleprobe detection system is described in U.S. Pat. No. 4,581,333 herebyincorporated by reference in its entirety.

In addition, automation of a nucleic acid detection system targetingunamplified or amplified nucleic acid, or a combined automatedamplification/detection system will generally be adaptable to the use ofnucleic acid capture oligonucleotides that are attached to some form ofsolid support system. Examples of such attachment and methods forattachment of nucleic acid to solid support are found in U.S. Pat. No.5,489,653 and 5,510,084 both of which are hereby incorporated byreference.

Automation of amplification, detection, and a combination ofamplification and detection is desirable to reduce the requirement ofmultiple user interactions with the assay. Apparatus and methods foroptically analyzing test materials are described for example in U.S.Pat. No. 5,122,284 (hereby incorporated by reference in its entirety).Automation is generally believed to be more economical, efficient,reproducible and accurate for the processing of clinical assays. Thuswith the superior sensitivity and specificity of nucleic acid detectionassays, the use of amplification of nucleic acid sequences, andautomation at one or more phases of a assay protocol can enhance theutility of the assay protocol and its utility in a clinical setting.

Advantage of Internal Control Sequences

Nucleic acid amplification is highly sensitive to reaction conditions,and the failure to amplify and/or detect any specific nucleic acidsequences in a sample may be due to error in the amplification processas much as being due to absence of desired target sequence.Amplification reactions are notoriously sensitive to reaction conditionsand have generally required including control reactions with knownnucleic acid target and primers in separate reaction vessels treated atthe same time. However, internal control sequences added into the testreaction mixture would truly control for the success of theamplification process in the subject test reaction mixture and would bemost useful. U.S. Pat. No. 5,457,027 (hereby incorporated by referencein its entirety) teaches certain internal control sequences which areuseful as an internal oligonucleotide standard in isothermalamplification reactions for Mycobacterium tuberculosis.

However it would be extremely useful to have a general method ofgenerating internal control sequences, that would be useful as internalcontrols of the various amplification procedures, which are specificallytailored to be unaffected by the nucleic acid sequences present in thetarget organism, the host organism, or nucleic acids present in thenormal flora or in the environment. Generally, such internal controlsequences should not be substantially similar to any nucleic acidsequences present in a clinical setting, including human, pathogenicorganism, normal flora organisms, or environmental organisms which couldinterfere with the amplification and detection of the internal controlsequences.

Detection of More than one Nucleic Acid Sequence in a Single Assay

In general, a single assay reaction for the detection of nucleic acidsequences is limited to the detection of a single target nucleic acidsequence. This single target limitation increases costs and timerequired to perform clinical diagnostic assays and verification controlreactions. The detection of more than one nucleic acid sequence in asample using a single assay would greatly enhance the efficiency ofsample analysis and would be of a great economic benefit by reducingcosts, for example helping to reduce the need for multiple clinicalassays.

Multiple analyte detection in a single assay has been applied toantibody detection of analyte as in for example International PatentPublication number WO 89/00290 and WO 93/21346 both of which are herebyincorporated by reference in their entirety.

In addition to reducing cost, time required, the detection of more thanone nucleic acid target sequence in a single assay would reduce thechance of erroneous results. In particular multiple detection wouldgreatly enhance the utility and benefit using internal control sequencesand allow for the rapid validation of negative results.

SUMMARY OF THE INVENTION

The present invention comprises methods for the automated isothermalamplification and detection of a specific nucleic acid in a test sampleto be tested comprising:

a) combining a test sample to be tested with a buffer, a mixture of freenucleotides, specific oligonucleotide primers, and optionallythermostable nucleic acid polymerization enzyme, in a first reactionvessel and placing the reaction vessel in an automated apparatus suchthat;

b) the automated apparatus heats the first reaction vessel to atemperature, and for a time sufficient to denature, if necessary, thenucleic acid in the sample to be tested;

c) the automated apparatus cools the first reaction vessel to atemperature such that oligonucleotide primers can specifically anneal tothe target nucleic acid;

d) the automated apparatus transfers the reaction mixture from the firstreaction vessel to a second reaction vessel, and brings the reactionmixture in contact with themmolabile nucleic acid amplification enzyme;

e) the automated apparatus maintains the temperature of the secondreaction vessel at a temperature which allows primer mediatedamplification of the nucleic acid;

f) the automated apparatus contacts the amplified nucleic acid in thesecond reaction vessel with a capture nucleic acid specific for thenucleic acid to be tested such that they form a specifically-boundnucleic acid-capture probe complex;

g) the automated apparatus optionally washes the specifically capturedamplified nucleic acid such that non-specifically bound nucleic acid iswashed away from the specifically-bound nucleic acid-capture probecomplex;

h) the automated apparatus contacts the specifically-bound nucleicacid-capture probe complex with a labeled nucleic acid probe specificfor the amplified nucleic acid such that a complex is formed between thespecifically amplified nucleic acid and the labeled nucleic acid probe;

i) the automated apparatus washes the specifically-bound nucleicacid-capture probe-labeled probe complex such that non-specificallybound labeled probe nucleic acid is washed away from the specificallybound complex;

j) the automated apparatus contacts the specifically bound complex witha solution wherein an detection reaction between the labeled nucleicacid probe is effected between the solution and the label attached tothe nucleic acid such that a detectable signal is generated from thesample in proportion the amount of specifically-bound amplified nucleicacid in the sample;

 wherein the steps h, i, and j may occur sequentially or simultaneously;

k) the automated apparatus detects the signal and optionally displays avalue for the signal, or optionally records a value for the signal.

As used herein, the term test sample includes samples taken from livingpatients, from non-living patients, from surfaces, gas, vacuum orliquids, from tissues, bodily fluids, swabs from body surfaces orcavities, and any similar source. The term buffer as used hereencompasses suitable formulations of buffer which can support theeffective activity of a label, for example an enzyme placed into suchbuffer when treated at the appropriate temperature for activity andgiven the proper enzymatic substrate and templates as needed. The termspecific oligonucleotide nucleic acid primers means an oligonucleotidehaving a nucleic acid sequence which is substantially complementary toand will specifically hybridize/anneal to a target nucleic acid ofinterest and may optionally contain a promoter sequence recognized byRNA polymerase. The term reaction vessel means a container in which achemical reaction can be performed and preferably capable ofwithstanding temperatures of anywhere from about −80° C. to 100° C.

The instant invention further provides for the method described above,wherein the reaction buffer is a unified buffer and as such is suitablefor denaturation nucleic acids and annealing of nucleic acids, and isfurther capable of sustaining the enzymatic activity of nucleic acidpolymerization and amplification enzyme. Further encompassed by theinvention is the method wherein the nucleic acid amplification enzyme isadministered in the second reaction chamber as a single assay doseamount in a lyophilized pellet, and the reaction chamber is sealed priorto the amplification step.

The invention teaches an apparatus for the automated detection of morethan one nucleic acid target sequences or amplicons comprising a solidphase receptacle (SPR® pipet-like devise) coated with at least twodistinct zones of a capture nucleic acid oligonucleotide.

The invention teaches a method for the automated detection of more thanone nucleic acid target sequence comprising contacting a solid phasereceptacle (SPR® pipet-like devise) coated with at least two distinctcapture nucleic acid oligonucleotides in a single or multiple zones to asample to be tested and detecting a signal(s) from specifically boundprobe. In one embodiment of the invention, the SPR® is coated with twodistinct zones of capture nucleic acid oligonucleotides which arespecific for different nucleic acid sequence targets. In anotherembodiment of the invention, the SPR® is coated with at least onecapture probe for a target nucleic acid sequence, and one capture probefor an amplification control nucleic acid sequence which when detectedconfirms that amplification did take place.

The present invention also comprises an internal amplification positivecontrol nucleic acid having the nucleic acid sequence of RIC1 and asecond internal amplification positive control nucleic acid having thenucleic acid sequence of RIC2.

The present invention further comprises a method for generating aninternal amplification positive control nucleic acid consisting of:

generating random nucleic acid sequences of at least 10 nucleotides inlength, screening said random nucleic acid sequence and selecting forspecific functionality, combining in tandem a number of suchfunctionally selected nucleic acid sequences, and screening the combinednucleic acid sequence and optionally selecting against formation ofintra-strand nucleic acid dimers, or the formation of hairpinstructures.

BRIEF DESCRIPTION OF THE DRAWINGS

Presently preferred embodiments of the invention will be described inconjunction with the appended drawings, wherein like reference numeralsrefer to like elements in the various views, and in which:

FIG. 1 is a graph illustrating single dose reagent pellet temperaturestability;

FIG. 2 illustrates the general TMA protocol;

FIG. 3A is a schematic representation of a disposable dual chamberreaction vessel and the heating steps associated therewith to perform aTMA reaction in accordance with one possible embodiment of theinvention;

FIG. 3B is a schematic representation of alternative form of theinvention in which two separate reaction chambers are combined to form adual chamber reaction vessel;

FIG. 3C is a schematic representation of two alternative embodiments ofa dual chamber reaction vessel that are snapped into place in a teststrip for processing with a solid phase receptacle and optical equipmentin accordance with a preferred embodiment of the invention;

FIG. 4 is a schematic representation of an alternative embodiment of adual chamber reaction vessel formed from two separate chambers that arecombined in a manner to permit a fluid sample in one chamber to betransferred to the other chamber, with the combined dual chamber vesselplaced into a test strip such as illustrated in FIG. 3C;

FIG. 5 is a perspective view of a stand-alone amplification processingstation for the test strips having the dual chamber reaction vessels inaccordance with a presently preferred form of the invention;

FIG. 6 is a perspective view of one of the amplification modules of FIG.4/31, as seen from the rear of the module;

FIG. 7 is a perspective view of the front of the module of FIG. 5/32;

FIG. 8 is another perspective view of the module of FIG. 7;

FIG. 9 is a detailed perspective view of a portion of the test stripholder and 95° C. Peltier heating subsystems of the module of FIGS. 6-8;

FIG. 10 is an isolated perspective view of the test strip holder of FIG.9, showing two test strips installed in the test strip holder;

FIG. 11 is a detailed perspective view of the test strip holder or trayof FIG. 7;

FIG. 12 is a block diagram of the electronics of the amplificationprocessing station of FIG. 7;

FIG. 13 is a diagram of the vacuum subsystem for the amplificationprocessing station of FIG. 6; and

FIG. 14 is a graph of the thermal cycle of the station of FIG. 6.

FIG. 15 illustrates a schematic of the operation of the multiplex VIDASdetection.

FIG. 16 illustrates the production of SPR® with two distinct capturezones;

FIG. 17 illustrates the VIDAS apparatus strip configuration formultiplex detection;

FIG. 18 illustrates and graphs the results of verification of the VIDASmultiplex protocol detecting only NG target;

FIG. 19A/46A is a graph showing the results when 1×10¹² CT targets weremixed with 0, 1×10⁹, 1×10¹⁰, 1×10¹¹, or 1×10¹², NG targets, and detectedwith the VIDAS instrument using the multiplex protocol and SPRs coatedwith CT capture probes on the bottom zone of the SPR®, and NG captureprobes on the top zone of the SPR®.

FIG. 19B/46B illustrates the results when 1×10¹² NG targets was mixedwith 0, 1×10⁹, 1×10¹⁰, 1×10¹¹, or 1×10¹², NG targets, and detected withthe VIDAS instrument using the multiplex protocol and SPR® coated withCT capture probes on the bottom zone of the SPR®, and NG capture probeson the top zone of the SPR®.

FIG. 20A is a graph showing detection of Mtb nucleic acid by VIDASapparatus after amplification.

FIG. 20B is a graph showing detection of Mtb nucleic acid by VIDASapparatus.

FIG. 21 is a graph showing detection of Mtb nucleic acid by VIDASapparatus after amplification.

FIG. 22 is a graph showing detection of Mtb nucleic acid by VIDASapparatus after amplification using the binary/dual chamber protocol.

FIG. 23 illustrates the results generated by the method describedshowing a collection of strings of nucleic acid sequences and screeningfor specific functional parameters.

FIG. 24 shows the nucleic acid sequence of Random Internal Control 1(RIC1) with the possible oligonucleotide primers/probes foramplification and detection of the control sequence.

FIG. 25 shows an analysis of the possible secondary structuralcomponents of the RIC1 sequence.

FIG. 26 shows the nucleic acid sequence of Random Internal Control 2(RIC2) with the possible oligonucleotide primers/probes foramplification and detection of the control sequence.

FIG. 27 shows an analysis of the possible secondary structuralcomponents of the RIC2 sequence.

FIG. 28 illustrates results from detection of RIC1 DNA, where the ran21was the capture probe and ran33 was an enzyme-linked detector-probe, andshows that amplification and detection occurs under standard assayconditions.

FIG. 29 shows that RIC1 RNA, amplified by TMA and the chemicallyactivated signal detected on a VIDAS instrument (bioMérieux Vitek, Inc.)using the enzyme-linked detection system, has a limit of sensitivity ofabout 1000 molecules of RIC1 RNA (without optimization of conditions).

DESCRIPTION OF THE INVENTION

The following examples are provided to better illustrate certainembodiments of the present invention without intending to limit thescope of the invention.

EXAMPLE 1 Single Dose Reagents and Unified Buffer

The implementation of a TMA reaction (see U.S. Pat. No. 5,437,990)on-line in a VIDAS or off-line in a separate instrument (with detectionoccurring on a VIDAS instrument) requires modification of the chemistryused to perform the reaction manually. First, bulk packaged reagentsmust be modified into single aliquot doses, and second, the buffercomponents of the reaction has been altered to form a singlecomprehensive multifunctional unified buffer solution.

Under the current manual technology, the reagents are prepared aslyophilized “cakes” of multiple-assay quantities. The amplification andenzyme reagents thus must be reconstituted in bulk and aliquoted forindividual assays.

Thus the automated form of TMA on the VIDAS system improves on the abovemanual method by utilizing single dose pellets of lyophilized reactioncomponents that can be resuspended in a single unified buffer which willsupport sample dilution, denaturation of nucleic acids, annealing ofnucleic acids, and desired enzymatic activity.

A) Unified Buffer and Single Dose Reagents

To test the feasibility of single dose amplification reagents, standardChlamydia TMA Amplification and Enzyme reagents (Gen-Probe Inc.), thebulk reagents were reconstituted in 0.75 ml of water. 12.5 μl of eitherthe water reconstituted amplification or enzyme reagent (i.e. a singledose aliquots) were aliquoted into microcentrifuge tubes. These tubeswere placed in a vacuum centrifuge with low heat to remove water. Theend result of this procedure was microcentrifuge tube containing asmall, dry cake of either enzyme or amplification reagent at the bottomof the tube.

The combined Unified Buffer used in this example, consists of acombination of standard commercially available Gen-Probe Inc. SampleDilution Buffer (SDB), Amplification Reconstitution Buffer (ARB), andEnzyme Dilution Buffer (EDB) in a 2:1:1 ratio. To each driedamplification reagent microfuge tube was added 100 μl of the combinedUnified Buffer, and positive control nucleic acid (+), and overlaid with100 μl of silicone oil. The tube was then heated to 95° C. for 10minutes and then cooled to 42° C. for 5 minutes. The 200 μl total volumewas then transferred to a tube containing the dried enzyme reagent. Thiswas then gently mixed to resuspend the enzyme reagent, and the solutionwas heated for one hour at 42° C.

Control reactions were prepared using Gen-Probe Control reagents whichwere reconstituted in the normal 1.5 ml of ARB or EDB according toinstructions provided in the Gen-Probe kit. In each control reaction 25μl of the reconstituted amplification reagent was combined with 50 μl orthe SDB with the positive control nucleic acid (+). The mixture was alsoheated to 95° C. for 10 minutes and then cooled to 42° C. for 5 minutes.To this was added 25 μl of the reconstituted enzyme reagent andincubated at 42° C. for one hour. Negative control had no nucleic acid.

Both the test Unified Buffer (Unified) reactions and the standardControl 15 (Control) reactions were then subjected to the Gen-Probe Inc.standard Hybridization Protection Assay (HPA) protocol. Briefly, 100 μlof a Chlamydia trachomatis specific nucleic acid probe was added to eachtube and allowed to hybridize for 15 minutes at 60° C. Then 300 μl ofSelection Reagent was added to each tube and the differential hydrolysisof hybridized and unhybridized probe was allowed to occur for 10minutes. The tubes were then read in a Gen-Probe Inc. Leader 50luminometer and the resultant data recorded as Relative Light Units(RLU) detected from the label, as shown in Table 1 below. Data reportedas RLU, standard C. Trachomatis TMA/HPA reaction.

TABLE 1 Unified single dose aliquot of amplification and enzyme reagentsControl (+) Unified (+) Control (−) Unified (−) 2,264,426 2,245,4956,734 3,993 2,156,498 2,062,483 3,484 3,765 1,958,742 2,418,531 5,4395,836 2,451,872 2,286,773 2,346,131 1,834,198

The data in Table 1 demonstrates that comparable results are obtainedwhen using the single dose aliquots of dried amplification and enzymereagent. In addition, the data shows that the results were comparableusing three separate buffers (ARB, EDB and SDB) and one unified combinedbuffer (SDB, ARB and EDB combined at a ratio of 2:1:1) to resuspend thereagents and run the reactions.

B) Pellitization of Single Dose Reagents

In order to simplify the single dose aliquoting of reagents, methodswhich will allow for pelletization of these reagents in single dosealiquots were used. Briefly, reagent pellets (or beads) can be made byaliquoting an aqueous solution of the reagent of choice (that has beencombined with an appropriate excipient, such as D(+) Trehalose(α-D-Glucopyranosyl-α-D-glucopyranoside, purchased from PfanstiehlLaboratories, Inc., Waukegan, Ill.) into a cryogenic fluid, and thenusing sublimation to remove the water from the pellet. Once thereagent/trehalose mixture is aliquoted into the cryogenic fluid, itforms a spherical frozen pellet. These pellets are then placed in alyophilizer where the frozen water molecules sublimate during the vacuumcycle. The result of this procedure is small, stable, non-flakingreagent pellets which can be dispensed into the appropriate packaging.Single dose aliquot pellets of reagents which contained RT, T7 and sugarwere subjected to a wide range of temperatures to examine pelletstability. After being subject to a test temperature for 10 minutes, thepellets were then used for CT amplification. The results are graphed inFIG. 1. The results show that the single dose reagent pellet remainsstable even after to exposure to high temperatures for 10 minutes.

The extraordinary stability of enzymes dried in trehalose has beenpreviously reported (Colaco et al., 1992, Bio/Technology, 10, 1007)which has renewed interest in research on long-term stabilization ofproteins has become a topic of interest (Franks, 1994, Bio/Technology,12, 253). The resulting pellets of the amplification reagent and enzymereagents were tested by use in C. Trachomatis TMA/PA reactions.

The prepared amplification pellets were placed in a tube to which wasadded 75 μl of a mixture of ARB and SDB (mixed in a 1:2 ratio) withpositive control nucleic acid. This sample was then heated to 95° C. for10 minutes and then cooled to 42° C. for 5 minutes. To this was added 25μl of enzyme reagent, which had been reconstituted using standardGen-Probe Inc. procedure. This mixture was allowed to incubate for onehour at 42° C. The reactions were then analyzed by the HPA procedure, asdescribed above. The results of this test are reported as RLU in Table2, and labeled AMP Pellets(+). As above, negative control reactions wererun without nucleic acid (−).

The prepared enzyme pellets were tested by heating 100 μl of acombination of SDB with positive control nucleic acid, EDB, and thestandard reconstituted amplification reagent (in a 2:1:1 ratio) at 95°C. for 10 minutes and then cooled to 42° C. for 5 minutes. The totalvolume of the reaction mix was added to the prepared enzyme pellet.After the pellet was dissolved, the reaction was heated to 42° C. forone hour and then subjected to HPA analysis as above. The results ofthis test are reported as RLU in Table 2 below, labeled Enzyme Pellet(+). Control reactions were prepared using standard Gen-Probe Inc.reagents following standard procedure. Data reported as RLU, standard C.Trachomatis TMA/HPA reaction.

TABLE 2 Single dose aliquot of pelleted amplification and enzymereagents Amp Pellets Amp Pellets Enzyme Enzyme Control (+) (+) (−)Pellets (+) Pellets (−) 2,363,342 2,451,387 2,619 2,240,989 3,4182,350,028 2,215,235 2,358 3,383,195 1,865 2,168,393 2,136,645 3,4212,596,041 2,649 2,412,876 2,375,541 2,247 2,342,288 1,653

The data in Table 2 demonstrates that there was no significantdifference when using the standard Gen-Probe Inc. reagents, or thedried, prepared, single dose amplification reagent pellet, or the enzymereagent pellet. Thus the single dose aliquots of reagents are suitablefor use with a single unified buffer for application to automation usinga VIDAS system.

EXAMPLE 2 Automated Isothermal Amplification Using Thermolabile Enzymes

In order to automate the isothermal amplification assay reaction for usewith clinical assay apparatus, such as a VIDAS instrument (BioMérieuxVitek, Inc.), a novel dual-chamber reaction vessel has been designed toimplement the use of the unified buffer and single reaction aliquotreagent pellets described above in isothermal amplification assay oftest samples which can be further used in combination with a stand aloneprocessing station.

A) Dual Reaction Chambers

The use of two chambers will facilitate keeping separate the heat stablesample/amplification reagent (containing the specific primers andnucleotides) from the heat labile enzymatic components (i.e. RNA reversetranscriptase, RNA polymerase RNase H). FIG. 3A is a schematicrepresentation of a disposable dual chamber reaction vessel 10 and theheating steps associated therewith to perform a TMA reaction inaccordance with one possible embodiment of the invention. Chamber Acontains the amplification mix, namely deoxynucleotides, primers, MgCl₂and other salts and buffer components. Chamber B contains theamplification enzyme that catalyzes the amplification reaction, e.g., T7and/or RT. After addition of the targets (or patient sample) intochamber A, heat is applied to chamber A to denature the DNA nucleic acidtargets and/or remove RNA secondary structure. The temperature ofchamber A is then cooled down to allow primer annealing. Subsequently,the solution of chamber A is brought into contact with chamber B.Chambers A and B, now in fluid communication with each other, are thenmaintained at the optimum temperature for the amplification reaction,e.g., 42 degrees C. By spatially separating chamber A from chamber B,and applying the heat for denaturation to chamber A only, thethermolabile enzymes in chamber B are protected from inactivation duringthe denaturation step.

FIG. 3B is a schematic representation of an alternative form of theinvention in which two separate reaction chambers 12 and 14 are combinedto form a dual chamber reaction vessel 10. Like the embodiment of FIG.3A, Chamber A is pre-loaded during a manufacturing step with anamplification mix, namely nucleotides, primers, MgCl₂ and other saltsand buffer components. Chamber B is pre-loaded during manufacturing withthe amplification enzyme that catalyzes the amplification reaction,e.g., T7 and/or RT. Fluid sample is then introduced into chamber A. Thetargets are heated for denaturation to 95° C. in chamber A. Aftercooling chamber A to 42° C., the solution in chamber A is brought intocontact with the enzymes in chamber B to trigger the isothermalamplification reaction.

If the reaction vessel is designed such that, after having brought thecontents of chambers A and B into contact, the amplification chamberdoes not allow any exchange of materials with the environment, a closedsystem amplification is realized that minimizes the risk ofcontaminating the amplification reaction with heterologous targets oramplification products from previous reactions.

FIG. 3C is a schematic representation of two alternative dual chamberreaction vessels 10 and 10′ that are snapped into place in a test strip19 for processing with a solid phase receptacle and optical equipment inaccordance with a preferred embodiment of the invention. In theembodiments of FIG. 3, a unidirectional flow system is provided. Thesample is first introduced into chamber A for heating to thedenaturation temperature. Chamber A contains the dried amplificationreagent mix 16. After cooling, the fluid is transferred to chamber Bcontaining the dried enzyme 18 in the form of a pellet. Chamber B ismaintained at 42° C. after the fluid sample is introduced into ChamberB. The amplification reaction takes place in Chamber B at the optimumreaction temperature (e.g., 42° C.). After the reaction is completed,the test strip 19 is then processed in a machine such as the VIDAS®instrument available from bioMérieux Vitek, Inc., the assignee of thepresent invention. Persons of skill in the art are familiar with theVIDAS® instrument.

The steps of heating and cooling of chamber A could be performed priorto the insertion of the dual chamber disposable reaction vessel 10 or10′ into the test strip 16, or, alternatively, suitable heating elementscould be placed adjacent to the left hand end 24 of the test strip 19 inorder to provide the proper temperature control of the reaction chamberA. The stand alone amplification processing station of FIGS. 4-14,described below, incorporates suitable heating elements and controlsystems to provide the proper temperature control for the reactionvessel 10.

FIG. 4 is a schematic representation of an alternative embodiment of adual chamber reaction vessel 10″ formed from two separate interlockingvessels 10A and 10B that are combined in a manner to permit a fluidsample in one chamber to flow to the other, with the combined dualchamber vessel 10″ placed into a test strip 19 such as described abovein FIG. 3A. The fluid sample is introduced into chamber A, whichcontains the dried amplification reagent mix 16. Vessel A is then heatedoff-line to 95 degrees C, then cooled to 42 degrees C. The two vessels Aand B are brought together by means of a conventional snap fit betweencomplementary locking surfaces on the tube projection 26 on chamber Band the recessed conduit 28 on chamber A. The mixing of the samplesolution from chamber A with the enzyme from chamber B occurs since thetwo chambers are in fluid communication with each other, as indicated bythe arrow 30. The sample can then be amplified in the combined dualchamber disposable reaction vessel 10″ off-line, or on-line by snappingthe combined disposable vessel 10″ into a modified VIDAS® strip. TheVIDAS® instrument could perform the detection of the amplificationreaction in known fashion.

B) Amplification Station

FIG. 5 is a perspective view of a stand-alone amplification processingsystem 200 for the test strips 19 having the dual chamber reactionvessels in accordance with a presently preferred form of the invention.The system 200 consists of two identical amplification stations 202 and204, a power supply module 206, a control circuitry module 208, a vacuumtank 210 and connectors 212 for the power supply module 206. The tank210 has hoses 320 and 324 for providing vacuum to amplification stations202 and 204 and ultimately to a plurality of vacuum probes (one perstrip) in the manner described above for facilitating transfer of fluidfrom the first chamber to the second chamber. The vacuum subsystem isdescribed below in conjunction with FIG. 14.

The amplification stations 202 and 204 each have a tray for receiving atleast one of the strips and associated temperature control, vacuum andvalve activation subsystems for heating the reaction wells of the stripto the proper temperatures, transferring fluid from the first chamber inthe dual chamber reaction wells to the second chamber, and activating avalve such as a thimble valve to open the fluid channel to allow thefluid to flow between the two chambers.

The stations 202 and 204 are designed as stand alone amplificationstations for performing the amplification reaction in an automatedmanner after the patient or clinical sample has been added to the firstchamber of the dual chamber reaction vessel described above. Theprocessing of the strips after the reaction is completed with an SPRtakes place in a separate machine, such as the VIDAS® instrument.Specifically, after the strips have been placed in the stations 202 and204 and the reaction run in the stations, the strips are removed fromthe stations 202 and 204 and placed into a VIDAS® instrument forsubsequent processing and analysis in known fashion.

The entire system 200 is under microprocessor control by anamplification system interface board (not shown in FIG. 5). The controlsystem is shown in block diagram form in FIG. 12 and will be describedlater.

Referring now to FIG. 6, one of the amplification stations 202 is shownin a perspective view. The other amplification station is of identicaldesign and construction. FIG. 7 is a perspective view of the front ofthe module of FIG. 6.

Referring to these figures, the station includes a vacuum probe slidemotor 222 and vacuum probes slide cam wheel 246 that operate to slide aset of vacuum probes 244 (shown in FIG. 7) for the thimble valves up anddown relative to a vacuum probes slide 246 to open the thimble valvesand apply vacuum so as to draw the fluid from the first chamber of thereaction vessel 10 to the second chamber. The vacuum probes 244reciprocate within annular recesses provided in the vacuum probes slide246. Obviously, proper registry of the pin structure and vacuum probe244 with corresponding structure in the test strip as installed on thetray needs to be observed.

The station includes side walls 228 and 230 that provide a frame for thestation 202. Tray controller board 229 is mounted between the side walls228 and 230. The electronics module for the station 202 is installed onthe tray controller board 229.

A set of tray thermal insulation covers 220 are part of a thermalsubsystem and are provided to envelop a tray 240 (FIG. 7) that receivesone or more of the test strips. The insulation covers 220 help maintainthe temperature of the tray 240 at the proper temperatures. The thermalsubsystem also includes a 42° C. Peltier heat sink 242, a portion ofwhich is positioned adjacent to the second chamber in the dual chamberreaction vessel in the test strip to maintain that chamber at the propertemperature for the enzymatic amplification reaction. A 95° C. heat sink250 is provided for the front of the tray 240 for maintaining the firstchamber of the reaction well in the test strip at the denaturationtemperature.

FIG. 8 is another perspective view of the module of FIG. 7, showing the95° C. heat sink 250 and a set of fins 252. Note that the 95° C. heatsink 250 is positioned to the front of and slightly below the tray 240.The 42° C. heat sink 242 is positioned behind the heat sink 250.

FIG. 9 is a detailed perspective view of a portion of the tray 240 thatholds the test strips (not shown) as seen from above. The tray 240includes a front portion having a base 254, a plurality of discontinuousraised parallel ridge structures 256 with recessed slots 258 forreceiving the test strips. The base of the front 254 of the tray 240 isin contact with the 95° C. heat sink 250. The side walls of the parallelraised ridges 256 at positions 256A and 256B are placed as close aspossible to the first and second chambers of the reaction vessel 10 ofFIG. 3A so as to reduce thermal resistance. The base of the rear of thetray 240 is in contact with a 42° C. Peltier heat sink, as best seen inFIG. 8. The portion 256B of the raised ridge for the rear of the tray isphysically isolated from portion 256A for the front of the tray, andportion 256B is in contact with the 42° C. heat sink so as to keep thesecond chamber of the reaction vessel in the test strip at the propertemperature.

Still referring to FIG. 9, the vacuum probes 244 include a rubber gasket260. When the vacuum probes 244 are lowered by the vacuum probe motor222 (FIG. 6) the gaskets 260 are positioned on the upper surface of thetest strip surrounding the vacuum port in the dual chamber reactionvessel so as to make a tight seal and permit vacuum to be drawn on thesecond chamber.

FIG. 10 is an isolated perspective view of the test strip holder or tray240 of FIG. 9, showing two test strips installed in the tray 240. Thetray 240 has a plurality of lanes or slots 241 receiving up to six teststrips 19 for simultaneous processing. FIG. 10 shows the heat sinks 242and 250 for maintaining the respective portions of the tray 240 andridges 256 at the proper temperature.

FIG. 11 is a detailed perspective view of the test strip holder or tray240 as seen from below. The 95° C. Peltier heat sink which would bebelow front portion 254 has been removed in order to better illustratethe rear heat sink 242 beneath the rear portion of the tray 240.

FIG. 12 is a block diagram of the electronics and control system of theamplification processing system of FIG. 5. The control system is dividedinto two boards 310 and 311, section A 310 at the top of the diagramdevoted to amplification module or station 202 and the other board 311(section B) devoted to the other module 204. The two boards 310 and 311are identical and only the top section 310 will be discussed. The twoboards 310 and 311 are connected to an amplification station interfaceboard 300.

The interface board 300 communicates with a stand alone personalcomputer 304 via a high speed data bus 302. The personal computer 304 isa conventional IBM compatible computer with hard disk drive, videomonitor, etc. In a preferred embodiment, the stations 202 and 204 areunder control by the interface board 300.

The board 310 for station 202 controls the front tray 240 which ismaintained at a temperature of 95° C. by two Peltier heat sink modules,a pair of fans and a temperature sensor incorporated into the frontportion 254 of the tray 240. The back of the tray is maintained at atemperature of 42° C. by two Peltier modules and a temperature sensor.The movement of the vacuum probes 244 is controlled by the probes motor222. Position sensors are provided to provide input signals to the traycontroller board as to the position of the vacuum probes 244. The traycontroller board 310 includes a set of drivers 312 for the active andpassive components of the system which receive data from the temperatureand position sensors and issue commands to the active components, i.e.,motors, fans, Peltier modules, etc. The drivers are responsive tocommands from the amplification interface board 300. The interface boardalso issues commands to the vacuum pump for the vacuum subsystem, asshown.

FIG. 13 is a diagram of the vacuum subsystem 320 for the amplificationprocessing stations 202 and 204 of FIG. 5. The subsystem includes a 1liter plastic vacuum tank 210 which is connected via an inlet line 322to a vacuum pump 323 for generating a vacuum in the tank 210. A vacuumsupply line 324 is provided for providing vacuum to a pair of pinchsolenoid valves 224 (see FIG. 6) via supply lines 324A and 324B. Thesevacuum supply lines 324A and 324B supply vacuum to a manifold 226distributing the vacuum to the vacuum probes 244. Note the pointed tips245 of the vacuum probes 244 for piercing the film or membrane 64covering the strip 19. The vacuum system 320 also includes adifferential pressure transducer 321 for monitoring the presence ofvacuum in the tank 210. The transducer 321 supplies pressure signals tothe interface board 300 of FIG. 12.

FIG. 14 is a representative graph of the thermal cycle profile of thestation of FIG. 5. As indicated in line 400, after an initial ramp up402 in the temperature lasting less than a minute, a first temperatureT1 is reached (e.g., a denaturation temperature) which is maintained fora predetermined time period, such as 5-10 minutes, at which time areaction occurs in the first chamber of the reaction vessel. Thereafter,a ramp down of temperature as indicated at 404 occurs and thetemperature of the reaction solution in the first chamber of thereaction vessel 10 cools to temperature T2. After a designated amount oftime after cooling to temperature T2, a fluid transfer occurs in whichthe solution in the first chamber is conveyed to the second chamber.Temperature T2 is maintained for an appropriate amount of time for thereaction of interest, such as one hour. At time 406, the temperature israised rapidly to a temperature T3 of 65° C. to stop the amplificationreaction. For a TMA reaction, it is important that the ramp up time fromtime 406 to time 408 is brief, that is, less than 2 minutes andpreferably less than one minute. Preferably, all the ramp up and rampdown of temperatures occur in less than a minute.

Other embodiments of reaction vessels and amplification stationcomponents are also envisioned, and certain examples of such alternativeembodiments are described in copending U.S. patent application of LuigiCatanzariti et al., serial no. hereby incorporated by reference in theentirety.

EXAMPLE 3 Automated VIDAS Test for Non-amplified and Amplified Detectionof Mycobacterium tuberculosis (Mtb)

Using the VIDAS instrument (BioMérieux Vitek, Inc.), modified to 42° C.,we have developed an in-line simple rapid nucleic acid amplification anddetection assay for the clinical laboratory for the detection of Mtb intest samples which can be completed in a short time. The entire assay isdesigned to take place on a single test strip, minimizing the potentialfor target or amplicon contamination. The amplification based assay iscapable of detection of Mtb where the sample contains only 5 cellssimilar to the sensitivity achieved by the Gen-Probe commercial kit.

The amplification based assay utilizes isothermal transcription-mediatedamplification (TMA) targeting unique sequences of rRNA, followed byhybridization and enzyme-linked fluorescent detection of nucleic acidprobe in the VIDAS instrument.

The amplification/detection assay can detect approximately 1 fg of MtbrRNA, or less than one Mtb organism per test, and is specific for allmembers of the Mtb complex. Specific probes for the detection of Mtb canbe found in C. Mabilat, 1994, J. Clin. Microbiol. 32, 2707.

Standard smears for acid-fast bacilli are not always reliable as adiagnostic tool, and even when positive may be a mycobateria other thanMtb. Currently, standard methods for diagnosis of tuberculosis requiresculturing the slow-growing bacteria, and may take up to 6 weeks orlonger. During this time, the patient is usually isolated. Initialresults are that this automated test matches or exceeds the clinicalsensitivity of the culture method, and offers a highly sensitive methodto rapidly (in less than three hours) detect Mtb in infected samples,thereby aiding rapid diagnosis, isolation and treatment.

A) Sample Preparation

A 450 μl volume of specimen is added to 50 μl of specimen dilutionbuffer in a lysing tube containing glass beads, sonicated for 15 minutesat room temperature to lyse organisms, heat inactivated for 15 minutesat 95° C. Where required, isothermal S amplification was conducted asper a commercially available manual assay kit (Gen-Probe Inc.) followingthe kit instructions using standard kit reagents. However, similarassays can be conducted using the modified components as described inthe Examples above.

B) Detection

In order for the automated detection assay to operate, the detectionsystem requires hybridization of the target nucleic acid or amplicon toa specific capture nucleic acid bound to a solid support, (in the VIDASsystem called a “solid phase receptacle” SPR® pipet-like devise), and toa labeled detection probe nucleic acid (for example where the label canbe alkaline phosphatase, a chemiluminescent signal compound, or otherreagent that will allow for specific detection of bound probe).

In an automated system such as the VIDAS, after several wash steps toremove unbound probe, the SPR® transfers the probe-target hybrid to anenzyme substrate, whereby the detectable signal is triggered from thebound probe and detected by the assay instrument. In one embodiment, theprobe is conjugated to alkaline phosphatase, and once placed in contactwith substrate of methyl umbelliferyl phosphate (MUMP), the substrate isconverted into 4-methyl umbelliferone (4-MU) by the alkalinephosphatase. The 4-MU produces fluorescence which is measured andrecorded by the standard VIDAS instrument as relative fluorescence units(RFU). When target nucleic acid is not present, no probe is bound, andno substrate is converted, thus no fluorescence is detected.

C) Analytical Sensitivity: Controls

Generally controls are prepared in a matrix of specimen dilution bufferwith positive controls containing 5 fg of Mtb rRNA, or the equivalentrRNA of approximately 1 M. tb cell. Sensitivity of the automated probeassay can be determined by testing dilutions of lysed M tb cells. Thecell lysates can generally be prepared with a 1 μl loop of cells (theassumption being that there are approximately 1×10⁹ colony forming units(CFU) per 1 μl loop-full, based upon previous titration and CFUexperiments). Dilutions of the Mtb lysates can then be tested with theautomated probe assay.

FIG. 20A is a graph showing detection of Mtb amplicons according to theGen-Probe kit. FIG. 20B is a graph showing detection of Mtb ampliconsfrom the same reactions as in FIG. 20A by the VIDAS instrument.

FIG. 21 is a graph showing amplification and detection of Mtb nucleicacids on to the modified VIDAS apparatus. Enzyme was used in liquid formand amplification was performed in-line with VIDAS assay instrument.

FIG. 22 is a graph showing amplification and detection of Mtb nucleicacids on the modified VIDAS apparatus using the binary/dual chamberdisposable reaction vessel. The denaturation step was performed off-lineof the VIDAS instrument, amplification and detection was performedin-line with VIDAS instrument.

EXAMPLE 4 Automated VIDAS Test for Amplified Detection of Chlamydiatrachomatis (CT)

Using the VIDAS instrument (BioMérieux Vitek, Inc.), we have developed asimple, fully automated, highly specific assay for the rapid detectionof Chlamydia trachomatis (CT) from test samples. The test utilizesisothermal TMA targeting unique sequences of the rRNA followed byhybridization and enzyme-linked fluorescence detection. The automatedtest specifically detects all the clinically important serovars ofChlamydia trachomatis (CT) from urogenital specimens in less than twohours. We obtained an analytical sensitivity of 0.5 fg of rRNA, or theequivalent of approximately {fraction (1/10)}^(th) of an elementary bodyof Chlamydia trachomatis (CT). Agreement between the automated test andGen-Probe's Amplified CT test for 207 clinical endocervical swabs andurines showed complete agreement. Chlamydia trachomatis (CT) infectionis the leading cause of sexually transmitted disease in the UnitedStates and Europe. It is currently estimated that about four million newCT infection occur each year in the United States.

Chlamydia trachomatis (CT) is a small obligate intracellular parasitethat causes infections in both females and males, adults and newborns.The greatest challenge to the control of CT infection is that as many as75% of infected women and 50% of infected men are asymptomatic. Thisresults in a large reservoir of unrecognized infected individuals whocan transmit the CT infection. The rapid and simple detection of CTinfection would greatly assist identification infected individuals.

A) Patient Specimens and Sample Preparation

Coded samples (207) were obtained from patients with symptoms consistentwith CT infection. The cervical samples were collected with a Gen-Probesample collection kit containing Gen-Probe transport medium; the urinesamples were collected into standard urine collection devices. Allsamples were stored at 4° C.

Cervical swabs were centrifuged at 425 ×g for 5 minutes to bring allliquid to the bottom of the tube. The swabs were then treated with 40 μlGen-Probe Specimen Preparation Reagent and incubated at 60° C. for 10minutes. 20 μl of the treated sample was then pipetted into 400 μl ofsample dilution buffer (SDB).

Two ml of each urine sample was warmed to 37° C. for 10 minutes andmicrofuged at 12,000 ×g for 5 minutes. The supernatant was discarded and300 μl of sample dilution buffer was added to each specimen. All 15serovars of CT were used for inclusive samples, specimens werequantified and 20 μl of specimens containing 4×10² LFU/ml (inclusionforming unit per ml) of each serovar was added to 400 μl of SDB. A panelof exclusive urogenital micororganisms was obtained and quantified and20 μl of 2×10⁹/ml microorganisms were pipetted into 400 μl of SDB.Positive control containing 0.5 fg rRNA or the equivalent of 0.1 CTelementary body was diluted in SDB.

B) Sample Amplification and VIDAS Detection

Samples were amplified using the TMA protocol, and rRNA targets werehybridized to oligomer conjugated to AMVE copolymer and an oligomerconjugated to alkaline phosphatase. See for example U.S. Pat. No.5,489,653 and 5,510,084. As described above, the solid phase receptacle(SPR® pipet-like devise) carries the hybrids through successive washsteps and finally into the substrate 4-MUP. The alkaline phosphataseconverts the substrate to fluoresence 4-MU, which is detected by theVIDAS assay machine and recorded as relative fluorescence units.

Table 2B below illustrates detection of CT by VIDAS automated assayfollowing amplification as RFV (RFV=RFU—Background RFU) againstconcentration of CT rRNA. Dilutions of C. trachomatis purified rRNA from0 to 200 molecules were amplified (n=3) and detected in the VIDASautomated probe assay. Detection limit is 20 molecules of purified rRNA.

TABLE 2B CT Detection by VIDAS rRNA Input Molecules VIDAS RFV 0 1 2 12120 3260 200 8487

C) Analytical Specificity and Results

Amplifications and detection were carried out in the presence of each ofthe following ATCC organisms with detections reported as RFV in Table 3below.

TABLE 3 Exclusivity panel for CT Bacillus subtilis Branhamella Candidaalbicans Chlamydia Chlamydia 33 catarrhalis 26 pneumoniae psittaci 15 3911 Escherichia coli Klebsiella Lactobacillus Neisseria Neisseria 11pneumoniae acidophilus elongata lactamica 13 27 44 18 NeisseriaNeisseria Propionibacterium Pseudomonas Staphylococcus meningitidis-Dmeningitidis-Y acnes aeruginosa aureus 61 52 14 13 13 StreptococcusStreptococcus Streptococcus Yersinia Chlamydia agalactiae bovispneumoniae enterolitica trachomatis 16 45 34 11 10673 Negative Control12

Analytical specificity for Chlamydia serovars data reported as RFV isshown in Table 4 below.

TABLE 4 Inclusivity Panel for CT Serovar A Serovar B Serovar Ba SerovarC Serovar D 5421 7247 9626 8066 10849 Serovar E Serovar F Serovar GSerovar H Serovar I 4608 9916 10082 7769 9733 Serovar J Serovar KSerovar L1 Serovar L2 Serovar L3 9209 2423 10786 1812 5883 PositiveNegative Control 3775 Control 9

Table 5 below illustrates the results of clinical cervical swab specimentesting for CT comparing results from the Gen-Probe manual AMP-CT assayand the VIDAS automated probe assay.

TABLE 5 Amplified Clinical Cervical Specimen Detection of CT Gen-Probemanual AMP-CT assay VIDAS off-line + − automated probe + 35  0 assay − 0 85

Table 6 below illustrates the results of clinical urine specimen testingcomparing the results of manual AMP-CT assay and the VIDAS automatedprobe assay.

TABLE 6 Amplified Clinical Urine Specimen detection of CT Gen-Probemanual AMP-CT assay VIDAS off-line + − automated probe + 25  0 assay − 0 62

Thus there was perfect agreement in assay results between the automatedprobe assay using the VIDAS instrument and the manual Gen-Probe AMP-CTassay.

EXAMPLE 5 Multiplex (Multiple Sequence) Nucleic Acid Detection

The value of diagnostic tests based on nucleic acid probes can besubstantially increased through the detection of multiple differentnucleic acid targets, and the use of internal positive controls. Anautomated method has been devised for use with the VIDAS instrument(BioMérieux Vitek, Inc.) which can discretely detect at least twodifferent nucleic acid target sequences in one assay reaction, and istermed the Multiplex protocol. Thus a nucleic acid amplificationprocedure, or a processed test sample may be screened for more than oneamplified nucleic acid target in the same assay. This method relies onthe spatial separation of discrete nucleic acid probes which can capturedifferent target nucleic acid sequences, on the Solid Phase Receptacle(SPR® pipet-like devise) of the VIDAS instrument. The SPR® is adisposable pipet-like tip which enables fluid movements as well asacting as the solid support for affinity capture. The multiplex captureby SPR® is demonstrated using capture probes specific for Chlamydiatrachomatis (CT) and Neisseria gonorrhoeae (NG).

FIG. 15 illustrates a schematic of the operation of the multiplex VIDASdetection. Solid phase receptacles (SPR® pipet-like devise) are coatedwith two distinct zones of oligonucleotides with nucleic acid sequenceswhich are used to specifically capture target nucleic acid sequenceswith their corresponding specific reporter probe nucleic acids labeledwith alkaline phosphatase (AKP). Following washes to remove unboundreporter probes, AKP localized to the SPR® bottom is detected with thefluorescent substrate 4-MUP. The AKP is stripped from the bottom of theSPR® with NaOH. The enzyme reaction well is emptied, washed, andre-filled with fresh 4-MUP. To ensure the removal of AKP from the bottomof the SPR®, the new substrate is exposed to the bottom of the SPR® andresidual fluorescence is measured. Finally, target bound to the top ofthe SPR® is detected by immersing the SPR® in the 4-MUP.

FIG. 16 illustrates the production of SPR® with two distinct capturezones. The SPR® is inserted tip-first into a silicon plug, which areheld in a rack. Differential pressure is used to uniformly draw a 1μg/ml solution of a specific capture probe, conjugated to AMVEcopolymer, into all SPR®s at one time. Attachment of the conjugate tothe SPR® surface is activated by passive adsorption for several hours atroom temperature. After washing, and drying, the SPR®s are capped with asmall adhesive disc and inserted into new racks in a tip-downorientation. The lower portion of the SPR® is then similarly coated witha second capture probe conjugate. SPR®s are stable when stored dry at 4°C.

FIG. 17 illustrates the VIDAS apparatus strip configuration formultiplex detection. The strip can be pre-filled with 200 μl ofAKP-probe mix (about 1×10¹² molecules each) in hybridization buffer inwell X1, 600 μl of wash buffer in wells X3, X4, X5, 600 μl of strippingreagent in wells X6 and X7, and 400 μl of AKP substrate in X8 and sealedwith foil. A foil-sealed optical cuvette (XA) containing 300 μl of 4-MUPis snapped into the strip, and the strips are inserted into the VIDASinstrument at 37° C. The multiplex VIDAS protocol is then executed usingSPR®s coated with two capture probes in distinct zones.

The VIDAS multiplex protocol can involve many steps. For example thevalidation test protocol contained 13 steps as follows:

1. Transfer 203 μl target from X0 to AKP-probes in X1,

2. Hybridize and capture to SPR®),

3. Wash SPR® (316 μl) twice with PBS/TWEEN (X3, X4),

4. 4-MUP to SPR® bottom (89.6 μl) in XA for 5.3 minutes then read,

5. 4-MUP to SPR® bottom (89.6 μl) in XA for 14.8 minutes then read,

6. Transfer used substrate from XA to X2 (5×67.1 μl),

7. Strip AKP from SPR® bottom (112.6 μl) with NaOH (X7),

8. Wash XA with fresh NaOH (3×112.6 μl; X6 to XA to X6),

9. Wash XA with PBS/TWEEN (3×112.6 μl; X5 to XA to X5),

10. Transfer fresh 4-MUP from X8 to XA (6×48 μl),

11. 4-MUP to SPR® bottom (89.6 μl) in XA for 10.7 minutes then read,

12. 4-MUP to SPR® top (294 μl) in XA for 5.5 minutes then read,

13. 4-MUP to SPR® top (294 μl) in XA for 15 minutes then read.

Hybridization, substrate, wash and stripping steps all involve multiplecycles of pipeting the respective solution into the SPR®, holding thesolution for a defined period of time, and pipeting the solution out ofthe SPR®. Hold times for hybridization, substrate and washing orstripping are 3.0, 0.5 and 0.17 minutes respectively. “Read” means thefluorescence is detected by the apparatus. Total assay time for theresearch protocol was about 1.75 hours but can be reduced to 75 minutes.

FIG. 18 illustrates and graphs the results of verification of the VIDASmultiplex protocol executed as described above, wherein the SPR® washomogeneously coated with only a single capture probe for Neisseriagonorrhoeae (NG). The number of NG oligonucleotide targets in the testsample was varied from 0, 1×10¹⁰, or 1×10¹¹ molecules in the testsample. The data shown are averages of replicate samples. The graph asillustrated is divided into two parts; the left and right halves showthe results of two fluorescent measurements from the lower and the upperzones of the SPR®, respectively. The measurements taken from the bottomzone after stripping the lower area of bound nucleic acid, and exposurefor about 11 minutes in fresh 4-MUP substrate was approximately 46 RFUfor all samples tested, and was equivalent to background fluorescencemeasured. This measurement is shown by the 0 time point in the center ofthe graph. Thus the graph illustrates two sequential sets ofmeasurements of fluorescence from a single SPR®, the first set ofmeasurements being taken from the bottom half of the

SPR® (left half of the graph), and a second set of measurements takenfrom the top of the SPR® (the right of the graph).

FIG. 19 illustrates Multiplex detection of CT and NG oligonucleotidetargets at different input amounts. FIG. 19A is a graph showing theresults when 1×10¹² CT targets were mixed with 0, 1×10⁹, 1×10¹⁰, 1×10¹¹,or 1×10¹², NG targets, and detected with the VIDAS instrument using themultiplex protocol and SPR®s coated with CT capture probes on the bottomzone of the SPR®, and NG capture probes on the top zone of the SPR®.FIG. 19B illustrates the results when 1×10¹² NG targets was mixed with0, 1×10⁹, 1×10¹⁰, 1×10¹¹, or 1×10¹², CT targets, and detected with theVIDAS instrument using the multiplex protocol and SPR®s coated with CTcapture probes on the bottom zone of the SPR®, and NG capture probes onthe top zone of the SPR®. The data is graphed as above where the graphillustrates two sequential sets of measurements of fluorescence from asingle SPR®, the first set of measurements being taken from the bottomhalf of the SPR® (left half of the graph), Stripped and verified (thecenter of the graph) and a second set of measurements taken from the topof the SPR® (the right of the graph).

Table 7 below summarizes the data obtained by Multiplex VIDAS detectionof CT and NG in a sample at various target levels, reported in RFUs.

TABLE 7 Detection of CT and NG targets in sample RFUs^(A) none^(B) 1 ×10⁹ 1 × 10¹⁰ 1 × 10¹¹ 1 × 10¹² 1 × 10¹³ none^(C) 43^(D)/40^(E) 43/11646/693 62/7116 174/11817 273/12136 1 × 10⁹ 189/41 246/118 169/773220/5750 422/12522 399/11401 1 × 10¹⁰ 1736/41 2258/125 1937/7341931/6639 2128/12390 2371/11180 1 × 10¹¹ 10339/48 9815/145 9858/7609369/4571 9784/11825 10252/10312 1 × 10¹² 12149/49 13520/148 12940/79613593/4397 11239/11786 10158/9900 1 × 10¹³ 11545/57 11713/121 10804/81512805/5404 12305/12326 11416/10490 ^(A)Data is reported in RFUs, after˜5 minute exposure of 4-MUP to bound AKP-probe ^(B)Columns are data forthat number of NG targets in sample ^(C)Rows are the data for thatnumber of CT targets in sample ^(D)The first value reported is RFUdetected from the CT assay portion ^(E)The second value reported is RFUdetected from the NG assay portion

Thus the multiplex VIDAS protocol is clearly operative and enables therapid and discrete detection of more than one different nucleic acidsignal in a sample. This protocol, and the SPR® coating can bemanipulated in many formats to present coating zones of differentsurface area with different sized gaps between detection zones. The SPR®can be coated with nucleic acids which are designed to capture differentregions of the same nucleic acid sequence to detect, for example,truncated gene expression, different alleles or alternatively splicedgenes. The SPR® can be coated to capture internal control nucleic acidsequences which can be used to detect and confirm successful nucleicacid amplification reactions. Thus the VIDAS protocol is a flexiblemethod for detection of more than one nucleic acid sequence in the samesample, in a single assay.

EXAMPLE 6 Internal Control Sequence and Method

The construction of internal control sequences composed of functionalbuilding blocks of sequences chosen by random generation of nucleic acidsequences for use as amplification reaction internal positive controlsideally requires that the control sequences be specifically designed tobe used for the various nucleic acid amplification protocols includingbut not limited to PCR, LCR, TMA, NASBA, and SDA. The internal controlnucleic acid sequence, in combination with the appropriate sequencespecific oligonucleotide primers or promoter-primers will generate apositive amplification signal if the amplification reaction wassuccessfully completed.

Ideally, the internal control nucleic acid is useful regardless of thenucleic acid sequences present in the target organism, the hostorganism, or nucleic acids present in the normal flora or in theenvironment. Generally, the internal control sequences should not besubstantially similar to any nucleic acid sequences present in aclinical setting, including human, pathogenic organism, normal floraorganisms, or environmental organisms which could interfere with theamplification and detection of the internal control sequences.

The internal control sequences of the instant invention are comprised offunctional blocks of sequences chosen from a list of randomly generatednucleic acid sequences. The functional blocks are segments which providefor a special property needed to allow for amplification, capture, anddetection of the amplification product. For example, in a TMA reaction,the internal control sequences are most useful when the functionalblocks meet certain functional requirements of the amplificationprotocol, such as: a) a primer binding site on the anti-sense strand; b)a capture site; c) a detector probe binding site; d) a T7-promotercontaining primer binding site on the sense strand. Each of thesefunctional elements has its own particular constraints, such as length,%G-C content, Tm, lack of homology to known sequences, and absence ofsecondary structural features (i.e. free from dimer formation or hairpinstructures) which can be used to select the appropriate sequence. Thusrandomly generated functional blocks of sequences can be screened forthe desired functional properties before use in constructing internalcontrol sequences.

In order to construct a internal control sequences having the desiredproperties comprising a specified number of functional blocks andsatisfying the desired constraints within each block, a random sequencegenerator was used to generate strings of numbers; each number beinglimited to the range from 0.000 to 4.000. The length of the strings isflexible and chosen based upon the desired lengths of the functionalblocks.

Each number in the string (i.e. n1, n2, n3, n4 . . . nx where x is thelength of the string) was then assigned a corresponding nucleotide asfollows: guanosine (G) if 0<n≦1; adenosine (A) if 1<n≦2; thymidine (T)if 2<n≦3; and cytosine (C) if 3<n≦4. A large collection of such stringswas produced and screened for those meeting the sequence and structuralrequirements of each functional block. FIG. 23 illustrates the resultsgenerated by the method described showing a collection of strings ofnucleic acid sequences and screening for specific functional parameters.

Potential internal control (IC) sequences were then constructed byassembling the functional blocks (selected at random) in the properorder. Finally, the assembled internal control sequences were thenexamined to insure that overall sequence and structural constraints weremaintained. For example, in a TMA internal control sequence the twoprimer binding sites should not have a significant base-pairingpotential or form stable 3′ dimer structures. Those internal controlsequences which pass thorough these layers of screening were thenphysically produced using overlapping oligonucleotides and tested forperformance in actual amplification/detection assays.

Although any one function block may have some homology to sequencespresent in a clinical setting (a perfect match of 21 nucleotide block isexpected at a random frequency of 1 in every 4e12 sequences or about4×10²¹; generated sequences were screened against GenBank data base) itis highly unlikely that all functional blocks will be found to havesubstantial homology. Since the internal control nucleic acid sequencesare constructed of a group of functional blocks placed in tandem, thechance possibility that a natural nucleic acid sequence will have anidentical string of nucleic acid sequence blocks in the same tandemorganization is remote.

Two specific internal control sequences have been constructed using themethod described above. Random Internal Control 1 (RIC1) is shown inFIG. 24 with the possible oligonucleotide primers/probes foramplification and detection of the control sequence. FIG. 25 shows ananalysis of the possible secondary structural components of the RIC1sequence. RIC1 was constructed using randomly generated strings ran16,ran 19, ran21 and ran33. The functional blocks requiring primer bindingwere met by ran16 and ran19, while the capture site was satisfied byran21 and the detector probe binding site was met by ran33.

Random Internal Control 2 (RIC2) is shown in FIG. 26 with the possibleoligonucleotide primers/probes for amplification and detection of thecontrol sequence. FIG. 27 shows an analysis of the possible secondarystructural components of the RIC2 sequence. Similarly to RIC1, RIC2 wasconstructed using randomly generated strings ran27, ran32, ran39 andran51. Thus, illustrating that it is also possible that the functionalblocks requiring primer binding, capture site, detector probe bindingsite can be met by alternative random sequences generated by the methoddescribed above. FIG. 28 illustrates results from detection of RIC1 DNA,where the ran21 was the capture probe and ran33 was an enzyme-linkeddetector-probe, and shows that detection occurs under standard assayconditions with expected fluorescence intensities. FIG. 29 shows thatRIC1 RNA, amplified by TMA and detected on a VIDAS instrument(BioMérieux Vitek, Inc.) using the enzyme-linked detection system, has alimit of sensitivity of about 1000 molecules of RIC1 RNA (withoutoptimization of conditions). Similar analysis of RIC2 sequences wasperformed and found to be similar to RIC1. It is significant that theamplification and detection system of the internal control functionedeffectively under the conditions optimized for the selected target.

As an alternative approach for multiplex detection using internalcontrols (IC), SPR®s can be homogeneously coated with a mixture ofdifferent capture nucleic acid sequences in a single, whole-SPR® zone.For example, two capture nucleic acid sequences can be combined in onezone, one specific for a target test sequence, and one specific for aninternal control sequence. Target amplicons, if present, and internalcontrol amplicons are simultaneously hybridized to the SPR® by thecapture probes. In the presence of labeled probe nucleic acid sequencesspecific for the target test nucleic acid sequence. Following washing, afirst read is done to so that the presence or absence of label on theSPR® is determined to ascertain the presence of the test target. Asecond hybridization is then done (sequential hybridization) to the SPR®using a detection label nucleic acid sequence specific for the internalcontrol. The SPR® is washed to remove excess unbound detection probe,and the second label is measured to indicate the presence or absence ofthe internal control. If the first signal is negative, a positive signalfrom the IC second read confirms the functionality of theamplification/detection system. In this case, one can conclude that thetest target nucleic acid sequence was truly absent (true negative). Ifthe first signal is positive, this alone is enough to confirmfunctionality of the amplification and detection system, and the secondsignal is immaterial (positive result). In the special case where thefirst the first and second label are the same, an additive signal willresult from the positive first read and the positive second IC read. Ifboth the first signal is negative and the second IC signal is alsonegative, then the amplification/detection functionality failed, whichcould be due to for example, sample interference or mechanical failure.In this case the test result is reported invalid (false negative) andre-testing is recommended.

There is great interest in the use of internal controls, the underlyingrational being that “. . . if the sample will not support theamplification of the internal control, it is unlikely to support theamplification of the target nucleic acid sequence.” (NCCLS DocumentMM3-A, Molecular Diagnostic Methods for Infectious Diseases; ApprovedGuideline, p. 55, March 1995).

Using a sequential hybridization approach with multiple detector probes,it has been possible to design protocols which allow for the discretedetection of first read signal (ie. pure CT signal) and an additive“mixed” second read (ie. additive CT and discrete signal for negatives;see Table 7A below). This protocol will not need stripping with NaOH.For example, Table 7A shows the results when different mixtures ofsynthetic targets were first captured with homogeneously coated SPR®s(CT and IC capture probes) and hybridized with the CT detector probe.After the first read, hybridization was performed with the IC detectorprobe, followed by a second read (same substrate).

This type of protocol can also be used for a combined GC/CT/intemalcontrol assay, if a screening approach is allowed (no discriminationbetween GC and/or CT positives during the first read). GC and CTspecific signals have to be resolved by running the CT and GC specificassays on screen positive samples (5-10% of cases, depending onprevalence) SPR®s would be coated homogeneously with 3 capture probes(CT/GC/internal control).

TABLE 7A Homogeneous Coated SPR ® Detection of multiple signals TargetCT 1^(st) Read IC 2^(nd) Read Bkg. RFU 10¹⁰ CT 7077 8608 58 10¹⁰ IC 584110 56 10¹⁰ IC/CT 5594 8273 57 10¹⁰ IC/CT 5712 8317 57 no target 66 8957

Thus internal control sequences described above are useful forapplication with VIDAS apparatus with coated SPR® and the use of theMultiplex system to provide for combined assay detection of a nucleicacid and monitoring control for successful reaction.

We claim:
 1. A device for the automated detection of at least two targetnucleic acid sequences, said device comprising a pipette-like devicehaving an internal and external surface and an internal cavity definedby said internal surface, wherein said internal surface is coated withat least two capture nucleic acid sequences which bind to said targetnucleic acid sequences to form hybridization complexes, and wherein saidtarget nucleic acids are discretely detected in said device.
 2. A deviceas in claim 1 wherein said capture nucleic acids are coated on saidinternal surface of said device in at least two distinct zones.
 3. Thedevice as claimed in claim 2 wherein said zones are spatially separated.4. The device as in claim 3 wherein said zones are coated homogenously.5. The device as in claim 2 wherein said zones are coated homogenously.6. A device as in claim 1 wherein said capture nucleic acids are coatedin said internal surface of said device in a single homogenous zone. 7.The device as in claim 1 wherein said capture nucleic acids are coatedon said internal surface of said device in two distinct zones.
 8. Thedevice as claimed in claim 7 wherein said zones are spatially separated.9. The device as in claims 1 wherein the internal cavity is coated witha first capture nucleic acid sequence and a second capture nucleic acidsequence, said first capture nucleic acid coated in a first zone andsaid second capture nucleic acid coated in a second zone of saidinternal cavity, wherein said first or second capture sequenceshybridize to complementary target nucleic acid sequences to formhybridization complexes in said first or second zone.
 10. The device asclaimed in claim 9 wherein said first and second zones are spatiallyseparated.
 11. The device as in claim 1 wherein said target nucleicacids are detected for results selected from the group consisting oftruncated gene expression, specific allele and specific spliced genes.